Abstract The objectives of this study are to understand the epigenetic roles of histone lysine crotonylation (Kcr) during late spermatogenesis and in paternal epigenetic inheritance. From the paternal germline to the next generation, epigenetic information is transmitted through compacted sperm chromatin. Mounting evidence suggests that the paternal epigenetic effects are mediated by histone post-translational modifications. However, the identity of histone marks and their roles in paternal epigenetic inheritance remains unclear. Additionally, it is largely unknown how histone marks are responsible for paternal epigenetic inheritance in response to exposure to environmental factors such as endocrine disrupters. We previously showed that lysine crotonylation is abundant during late spermatogenesis and is associated with X-linked active gene expression in postmeiotic spermatids. We also found that RNF8, an E3 ubiquitin ligase with a key role in the DNA damage response pathway, is responsible for the establishment of histone Kcr on the sex chromosomes during late spermatogenesis. Furthermore, Kcr is also associated with autosomal poised genes in postmeiotic spermatids, and these genes are activated after fertilization. These data raise the possibility that Kcr distinctively contributes to paternal epigenetic inheritance both on sex chromosomes and autosomes. In this proposal, we hypothesize that histone Kcr plays a key role in paternal epigenetic inheritance. To test this hypothesis, we have designed two aims. In Aim 1, we will identify and quantify the dynamic changes of histone Kcr sites by proteomics analyses and establish the reference Kcr epigenome in late spermatogenesis. We will generate the reference Kcr epigenome in response to exposure to endocrine disruptors in order to determine the depth of the effect. In Aim 2, we will analyze early embryonic stages after fertilization and determine whether histone Kcr regulates paternal epigenetic inheritance in response to exposure to endocrine disruptors. Collectively, our proteomics, ChIP-seq, and RNA-seq experiments will determine the depth of Kcr's involvement in paternal epigenetic inheritance.